Molecular dynamics of ribosomal elongation factors G and Tu

Translation on the ribosome is controlled by external factors. During polypeptide lengthening, elongation factors EF-Tu and EF-G consecutively interact with the bacterial ribosome. EF-Tu binds and delivers an aminoacyl-tRNA to the ribosomal A site and EF-G helps translocate the tRNAs between their binding sites after the peptide bond is formed. These processes occur at the expense of GTP. EF-Tu:tRNA and EF-G are of similar shape, share a common binding site, and undergo large conformational changes on interaction with the ribosome. To characterize the internal motion of these two elongation factors, we used 25 ns long all-atom molecular dynamics simulations. We observed enhanced mobility of EF-G domains III, IV, and V and of tRNA in the EF-Tu:tRNA complex. EF-Tu:GDP complex acquired a configuration different from that found in the crystal structure of EF-Tu with a GTP analogue, showing conformational changes in the switch I and II regions. The calculated electrostatic properties of elongation factors showed no global similarity even though matching electrostatic surface patches were found around the domain I that contacts the ribosome, and in the GDP/GTP binding region.     

Microcalorimetric study of elongation factor Tu from Thermus thermophilus in nucleotide-free,GDP and GTP forms and in the presence of elongation factor Ts

Elongation factor (EF) Tu undergoes profound nucleotide-dependent conformational changes in its functional cycle. The thermodynamic parameters of the different Thermus thermophilus EF-Tu forms, its domains I, II/III and III, were determined by microcalorimetry. Thermal transitions of the EF-Tu.GDP and EF-Tu.guanosine-5'-[beta,gamma-imido]triphosphate have a cooperative two-state character. Nucleotide removal affected the cooperativity of the thermal transition of EF-Tu. Microcalorimetric measurements of nucleotide-free EF-Tu and its separated domains showed that domains II/III have the main stabilizing role for the whole protein. Despite the fact that strong interactions between elongation factors Tu and Ts from T. thermophilus at 20 degrees C exist, the thermal transition of neither protein in the complex was significantly affected.     

Thiostrepton inhibition of tRNA delivery to the ribosome

Ribosome-stimulated hydrolysis of guanosine-5'-triphosphate (GTP) by guanosine triphosphatase (GTPase) translation factors drives protein synthesis by the ribosome. Allosteric coupling of GTP hydrolysis by elongation factor Tu (EF-Tu) at the ribosomal GTPase center to messenger RNA (mRNA) codon:aminoacyl-transfer RNA (aa-tRNA) anticodon recognition at the ribosomal decoding site is essential for accurate and rapid aa-tRNA selection. Here we use single-molecule methods to investigate the mechanism of action of the antibiotic thiostrepton and show that the GTPase center of the ribosome has at least two discrete functions during aa-tRNA selection: binding of EF-Tu(GTP) and stimulation of GTP hydrolysis by the factor. We separate these two functions of the GTPase center and assign each to distinct, conserved structural regions of the ribosome. The data provide a specific model for the coupling between the decoding site and the GTPase center during aa-tRNA selection as well as a general mechanistic model for ribosome-stimulated GTP hydrolysis by GTPase translation factors.     

Domain III of elongation factor G from Thermus thermophilus is essential for induction of GTP hydrolysis on the ribosome

Two elongation factors (EF) EF-Tu and EF-G participate in the elongation phase during protein biosynthesis on the ribosome. Their functional cycles depend on GTP binding and its hydrolysis. The EF-Tu complexed with GTP and aminoacyl-tRNA delivers tRNA to the ribosome, whereas EF-G stimulates translocation, a process in which tRNA and mRNA movements occur in the ribosome. In the present paper we report that: (a) intrinsic GTPase activity of EF-G is influenced by excision of its domain III; (b) the EF-G lacking domain III has a 10(3)-fold decreased GTPase activity on the ribosome, whereas its affinity for GTP is slightly decreased; and (c) the truncated EF-G does not stimulate translocation despite the physical presence of domain IV, which is also very important for translocation. By contrast, the interactions of the truncated factor with GDP and fusidic acid-dependent binding of EF-G.GDP complex to the ribosome are not influenced. These findings indicate an essential contribution of domain III to activation of GTP hydrolysis. These results also suggest conformational changes of the EF-G molecule in the course of its interaction with the ribosome that might be induced by GTP binding and hydrolysis.     

The importance of structural transitions of the switch II region for the functions of elongation factor Tu on the ribosome

Elongation factor Tu (EF-Tu) undergoes a large conformational transition when switching from the GTP to GDP forms. Structural changes in the switch I and II regions in the G domain are particularly important for this rearrangement. In the switch II region, helix alpha2 is flanked by two glycine residues: Gly(83) in the consensus element DXXG at the N terminus and Gly(94) at the C terminus. The role of helix alpha2 was studied by pre-steady-state kinetic experiments using Escherichia coli EF-Tu mutants where either Gly(83), Gly(94), or both were replaced with alanine. The G83A mutation slows down the association of the ternary complex EF-Tu.GTP.aminoacyl-tRNA with the ribosome and abolishes the ribosome-induced GTPase activity of EF-Tu. The G94A mutation strongly impairs the conformational change of EF-Tu from the GTP- to the GDP-bound form and decelerates the dissociation of EF-Tu.GDP from the ribosome. The behavior of the double mutant is dominated by the G83A mutation. The results directly relate structural transitions in the switch II region to specific functions of EF-Tu on the ribosome.     

Functionally significant mobile regions of Escherichia coli SecA ATPase identified by NMR

SecA, a 204-kDa homodimeric protein, is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. SecA promotes translocation by nucleotide-modulated insertion and deinsertion into the cytoplasmic membrane once bound to both the signal sequence and portions of the mature domain of the preprotein. SecA is proposed to undergo major conformational changes during translocation. These conformational changes are accompanied by major rearrangements of SecA structural domains. To understand the interdomain rearrangements, we have examined SecA by NMR and identified regions that display narrow resonances indicating high mobility. The mobile regions of SecA have been assigned to a sequence from the second of two domains with nucleotide-binding folds (NBF-II; residues 564-579) and to the extreme C-terminal segment of SecA (residues 864-901), both of which are essential for preprotein translocation activity. Interactions with ligands suggest that the mobile regions are involved in functionally critical regulatory steps in SecA.     

Delayed release of inorganic phosphate from elongation factor Tu following GTP hydrolysis on the ribosome

The dissociation of inorganic phosphate (P(i)) following GTP hydrolysis is a key step determining the functional state of many GTPases. Here, the timing of P(i) release from elongation factor Tu (EF-Tu) and its implications for the function of EF-Tu on the ribosome were studied by rapid kinetic techniques. It was found that P(i) release from EF-Tu is >20-fold slower than GTP cleavage and limits the rate of the conformational switch of EF-Tu from the GTP- to the GDP-bound form. The point mutation Gly94Ala in the switch 2 region of EF-Tu abolished the delay in P(i) release, suggesting that P(i) release is controlled by the mobility of the switch 2 region with Gly94 acting as a pivot. The rate of P(i) release or the conformational switch of EF-Tu does not affect the selection of aminoacyl-tRNA on the ribosome. Rather, the slow P(i) release may be a consequence of the tight interaction of the switch regions of EF-Tu with the gamma-phosphate and the ribosome in the GTPase activated state of the factor.     

Functional interaction between bases C1049 in domain II and G2751 in domain VI of 23S rRNA in Escherichia coli ribosomes

The factor-binding center within the Escherichia coli ribosome is comprised of two discrete domains of 23S rRNA: the GTPase-associated region (GAR) in domain II and the sarcin–ricin loop in domain VI. These two regions appear to collaborate in the factor-dependent events that occur during protein synthesis. Current X-ray crystallography of the ribosome shows an interaction between C1049 in the GAR and G2751 in domain VI. We have confirmed this interaction by site-directed mutagenesis and chemical probing. Disruption of this base pair affected not only the chemical modification of some bases in domains II and VI and in helix H89 of domain V, but also ribosome function dependent on both EF-G and EF-Tu. Mutant ribosomes carrying the C1049 to G substitution, which show enhancement of chemical modification at G2751, were used to probe the interactions between the regions around 1049 and 2751. Binding of EF-G-GDP-fusidic acid, but not EF-G-GMP-PNP, to the ribosome protected G2751 from modification. The G2751 protection was also observed after tRNA binding to the ribosomal P and E sites. The results suggest that the interactions between the bases around 1049 and 2751 alter during different stages of the translation process.     

The N-terminal region is crucial for the thermostability of the G-domain of Bacillus stearothermophilus EF-Tu

Bacterial elongation factor Tu (EF-Tu) is a model monomeric G protein composed of three covalently linked domains. Previously, we evaluated the contributions of individual domains to the thermostability of EF-Tu from the thermophilic bacterium Bacillus stearothermophilus. We showed that domain 1 (G-domain) sets up the basal level of thermostability for the whole protein. Here we chose to locate the thermostability determinants distinguishing the thermophilic domain 1 from a mesophilic domain 1. By an approach of systematically swapping protein regions differing between G-domains from mesophilic Bacillus subtilis and thermophilic B. stearothermophilus, we demonstrate that a small portion of the protein, the N-terminal 12 amino acid residues, plays a key role in the thermostability of this domain. We suggest that the thermostabilizing effect of the N-terminal region could be mediated by stabilizing the functionally important effector region. Finally, we demonstrate that the effect of the N-terminal region is significant also for the thermostability of the full-length EF-Tu.     

The Identification of the Determinants of the Cyclic, Sequential Binding of Elongation Factors Tu and G to the Ribosome

Experiments dedicated to gaining an understanding of the mechanism underlying the orderly, sequential association of elongation factor Tu (EF-Tu) and elongation factor G (EF-G) with the ribosome during protein synthesis were undertaken. The binding of one EF is always followed by the binding of the other, despite the two sharing the same—or a largely overlapping—site and despite the two having isosteric structures. Aminoacyl-tRNA, peptidyl-tRNA, and deacylated-tRNA were bound in various combinations to the A-site, P-site, or E-site of ribosomes, and their effect on conformation in the peptidyl transferase center, the GTPase-associated center, and the sarcin/ricin domain (SRD) was determined. In addition, the effect of the ribosome complexes on sensitivity to the ribotoxins sarcin and pokeweed antiviral protein and on the binding of EF-G•GTP were assessed. The results support the following conclusions: the EF-Tu ternary complex binds to the A-site whenever it is vacant and the P-site has peptidyl-tRNA; and association of the EF-Tu ternary complex is prevented, simply by steric hindrance, when the A-site is occupied by peptidyl-tRNA. On the other hand, the affinity of the ribosome for EF-G•GTP is increased when peptidyl-tRNA is in the A-site, and the increase is the result of a conformational change in the SRD. We propose that peptidyl-tRNA in the A-site is an effector that initiates a series of changes in tertiary interactions between nucleotides in the peptidyl transferase center, the SRD, and the GTPase-associated center of 23S rRNA; and that the signal, transmitted through a transduction pathway, informs the ribosome of the position of peptidyl-tRNA and leads to a conformational change in the SRD that favors binding of EF-G.     

Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure

Specific alterations of the elongation factor Tu (EF-Tu) polypeptide chain have been identified in a number of mutant species of this elongation factor. In two species, Ala-375, located on domain II, was found by amino acid analysis to be replaced by Thr and Val, respectively. These replacements substantially lower the affinity of EF-Tu.GDP for the antibiotic kirromycin. Since kirromycin can be cross-linked to Lys-357, also located on domain II but structurally very far from Ala-375, these data suggest that the replacements alter the relative position of domains I and II. The Ala-375 replacements also lower the dissociation rates of the binary complexes EF-Tu.GTP and the binding constants for EF-Tu.GTP and Phe-tRNA. It is conceivable that these effects are also mediated by movements of domains I and II relative to each other. Replacement of Gly-222 by Asp has been found in another mutant by DNA sequence analysis of the cloned tufB gene, coding for this mutant EF-Tu. Gly-222 is part of a structural domain, characteristic for a variety of nucleotide binding enzymes. Its replacement by Asp does not abolish the ability of EF-Tu to sustain protein synthesis. It increases the dissociation rate of EF-Tu.GTP by approximately 30%. In the presence of kirromycin this mutant species of EF-Tu.GDP does not bind to the ribosome, in contrast to its wild-type counterpart. A possible explanation is now open for experimental verification.     

Structure-based sequence alignment of elongation factors Tu and G with related GTPases involved in translation

The G domain and domain II in the crystal structure of Thermus thermophilus elongation factor G (EF-G) were compared with the homologous domains in Thermus aquaticus elongation factor Tu (EF-Tu). Sequence alignment derived from the structural superposition was used to define conserved sequence elements in domain II. These elements and previously known conserved sequence elements in the G domain were used to guide the alignment of the sequences of Sulfolobus acidocaldarius elongation factor 2, human elongation factor 2, and Escherichia coli initiation factor 2 and release factor 3 to the aligned sequences of EF-G and EF-Tu. This alignment, which deviates from previously published alignments, has evolutionary implications and leads to alternative interpretations of biochemical data concerning the interaction of elongation factors with the -sarcin/ricin region of the ribosome. A single conserved sequence motif in domain II was identified and used to further characterize the GTPase subfamily of translation factors and related proteins. It was shown that the motif is found in most if not all the members of the family. Apparently, the common characteristic of these GTPases is an extensive consensus structural unit that possibly accounts for a similar interaction with the ribosome and is composed of two domains homologous to the G domain and domain II in EF-Tu and EF-G.     

The structural and functional basis for the kirromycin resistance of mutant EF-Tu species in Escherichia coli.

A structural and functional understanding of resistance to the antibiotic kirromycin in Escherichia coli has been sought in order to shed new light on the functioning of the bacterial elongation factor Tu (EF-Tu), in particular its ability to act as a molecular switch. The mutant EF-Tu species G316D, A375T, A375V and Q124K, isolated by M13mp phage-mediated targeted mutagenesis, were studied. In this order the mutant EF-Tu species showed increasing resistance to the antibiotic as measured by poly(U)-directed poly(Phe) synthesis and intrinsic GTPase activities. The K'd values for kirromycin binding to mutant EF-Tu.GTP and EF-Tu.GDP increased in the same order. All mutation sites cluster in the interface of domains 1 and 3 of EF-Tu.GTP, not in that of EF-Tu.GDP. Evidence is presented that kirromycin binds to this interface of wild-type EF-Tu.GTP, thereby jamming the conformational switch of EF-Tu upon GTP hydrolysis. We conclude that the mutations result in two separate mechanisms of resistance to kirromycin. The first inhibits access of the antibiotic to its binding site on EF-Tu.GTP. A second mechanism exists on the ribosome, when mutant EF-Tu species release kirromycin and polypeptide chain elongation continues.     

Effects of domain exchanges between Escherichia coli and mammalian mitochondrial EF-Tu on interactions with guanine nucleotides, aminoacyl-tRNA and ribosomes.

Escherichia coli elongation factor (EF-Tu) and the corresponding mammalian mitochondrial factor, EF-Tumt, show distinct differences in their affinities for guanine nucleotides and in their interactions with elongation factor Ts (EF-Ts) and mitochondrial tRNAs. To investigate the roles of the three domains of EF-Tu in these differences, six chimeric proteins were prepared in which the three domains were systematically switched. E. coli EF-Tu binds GDP much more tightly than EF-Tumt. This difference does not reside in domain I alone but is regulated by interactions with domains II and III. All the chimeric proteins formed ternary complexes with GTP and aminoacyl-tRNA although some had an increased or decreased activity in this assay. The activity of E. coli EF-Tu but not of EF-Tumt is stimulated by E. coli EF-Ts. The presence of any one of the domains of EF-Tumt in the prokaryotic factor reduced its interaction with E. coli EF-Ts 2-3-fold. In contrast, the presence of any of the three domains of E. coli EF-Tu in EF-Tumt allowed the mitochondrial factor to interact with bacterial EF-Ts. This observation indicates that even domain II which is not in contact with EF-Ts plays an important role in the nucleotide exchange reaction. EF-Tsmt interacts with all of the chimeras produced. However, with the exception of domain III exchanges, it inhibits the activities of the chimeras indicating that it could not be productively released to allow formation of the ternary complex. The unique ability of EF-Tumt to promote binding of mitochondrial Phe-tRNAPhe to the A-site of the ribosome resides in domains I and II. These studies indicate that the interactions of EF-Tu with its ligands is a complex process involving cross-talk between all three domains.     

Conformational variability in Escherichia coli 70S ribosome as revealed by 3D cryo-electron microscopy

During protein biosynthesis, ribosomes are believed to go through a cycle of conformational transitions. We have identified some of the most variable regions of the E. coli 70S ribosome and its subunits, by means of cryo-electron microscopy and three-dimensional (3D) reconstruction. Conformational changes in the smaller 30S subunit are mainly associated with the functionally important domains of the subunit, such as the neck and the platform, as seen by comparison of heat-activated, non-activated and 50S-bound states. In the larger 50S subunit the most variable regions are the L7/L12 stalk, central protuberance and the L1-protein, as observed in various tRNA-70S ribosome complexes. Difference maps calculated between 3D maps of ribosomes help pinpoint the location of ribosomal regions that are most strongly affected by conformational transitions. These results throw direct light on the dynamic behavior of the ribosome and help in understanding the role of these flexible domains in the translation process.     

EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways

The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and P_i release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change.     

Codon-dependent conformational change of elongation factor Tu preceding GTP hydrolysis on the ribosome.

The mechanisms by which elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA to the A site of the ribosome and, in particular, how GTP hydrolysis by EF-Tu is triggered on the ribosome, are not understood. We report steady-state and time-resolved fluorescence measurements, performed in the Escherichia coli system, in which the interaction of the complex EF-Tu.GTP.Phe-tRNAPhe with the ribosomal A site is monitored by the fluorescence changes of either mant-dGTP [3'-O-(N-methylanthraniloyl)-2-deoxyguanosine triphosphate], replacing GTP in the complex, or of wybutine in the anticodon loop of the tRNA. Additionally, GTP hydrolysis is measured by the quench-flow technique. We find that codon-anticodon interaction induces a rapid rearrangement within the G domain of EF-Tu around the bound nucleotide, which is followed by GTP hydrolysis at an approximately 1.5-fold lower rate. In the presence of kirromycin, the activated conformation of EF-Tu appears to be frozen. The steps following GTP hydrolysis--the switch of EF-Tu to the GDP-bound conformation, the release of aminoacyl-tRNA from EF-Tu to the A site, and the dissociation of EF-Tu-GDP from the ribosome--which are altogether suppressed by kirromycin, are not distinguished kinetically. The results suggest that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements resulting in a conformational change of EF-Tu, possibly involving the effector region, which, in turn, triggers GTP hydrolysis.     

tRNA Dissociation from EF-Tu after GTP Hydrolysis: Primary Steps and Antibiotic Inhibition

In each round of ribosomal translation, the translational GTPase elongation factor Tu (EF-Tu) delivers a transfer RNA (tRNA) to the ribosome. After successful decoding, EF-Tu hydrolyzes GTP, which triggers a conformational change that ultimately results in the release of the tRNA from EF-Tu. To identify the primary steps of these conformational changes and how they are prevented by the antibiotic kirromycin, we employed all-atom explicit-solvent molecular dynamics simulations of the full ribosome-EF-Tu complex. Our results suggest that after GTP hydrolysis and P_i release, the loss of interactions between the nucleotide and the switch 1 loop of EF-Tu allows domain D1 of EF-Tu to rotate relative to domains D2 and D3 and leads to an increased flexibility of the switch 1 loop. This rotation induces a closing of the D1-D3 interface and an opening of the D1-D2 interface. We propose that the opening of the D1-D2 interface, which binds the CCA tail of the tRNA, weakens the crucial EF-Tu-tRNA interactions, which lowers tRNA binding affinity, representing the first step of tRNA release. Kirromycin binds within the D1-D3 interface, sterically blocking its closure, but does not prevent hydrolysis. The resulting increased flexibility of switch 1 explains why it is not resolved in kirromycin-bound structures.     

A signal relay between ribosomal protein S12 and elongation factor EF-Tu during decoding of mRNA

Codon recognition by aminoacyl-tRNA on the ribosome triggers a process leading to GTP hydrolysis by elongation factor Tu (EF-Tu) and release of aminoacyl-tRNA into the A site of the ribosome. The nature of this signal is largely unknown. Here, we present genetic evidence that a specific set of direct interactions between ribosomal protein S12 and aminoacyl-tRNA, together with contacts between S12 and 16S rRNA, provide a pathway for the signaling of codon recognition to EF-Tu. Three novel amino acid substitutions, H76R, R37C, and K53E in Thermus thermophilus ribosomal protein S12, confer resistance to streptomycin. The streptomycin-resistance phenotypes of H76R, R37C, and K53E are all abolished by the mutation A375T in EF-Tu. A375T confers resistance to kirromycin, an antibiotic freezing EF-Tu in a GTPase activated state. H76 contacts aminoacyl-tRNA in ternary complex with EF-Tu and GTP, while R37 and K53 are involved in the conformational transition of the 30S subunit occurring upon codon recognition. We propose that codon recognition and domain closure of the 30S subunit are signaled through aminoacyl-tRNA to EF-Tu via these S12 residues.     

Conformational changes in ribosomes upon interaction with elongation factors

Conformational changes in the ribosomes upon interaction with EF-Tu were studied by limited proteolysis with a set of proteases. The main results are: (1) The cleavage rate of S1 protein strongly depends on the cooperative effect of poly(U) and tRNA: (2) The conformation of L7/L12 proteins is modulated by interaction of elongation factors with the ribosome and depends on hydrolysis of GTP; (3) The sensitivity of some ribosomal proteins (S6, S7, S18, S19, L9, L16, L19, and L27) to proteases changes upon binding of EF-Tu and depends on the ribosome functional state in accordance with GTP hydrolysis. Most of these proteins are located far from the factor-binding center of the ribosome. The possible mechanism of conformational changes is discussed.